This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite the many excellent studies of EM morphology after RF/FSF into acetone, samples often suffer from ice crystal damage, which is detected in the higher resolution tomographic reconstructions. While for best results the initial vitrification of samples during rapid freezing is a crucial first step, the subsequent RF/FSF process may impose its own potential for ice damage. Due to the melting temperature of acetone at -94.9[unreadable]C, freeze substitution requires warming the specimen to -90[unreadable]C at the initiation of the substitution process which is significantly higher than the -140o C that marks the transition between vitrified and crystalline ice [1]. What counts for pure water may not be exactly the same for a mixed protein solution as the high solute concentration of the cytoplasm may raise the phase-transition point to some extent, but likely not to reach the melting temperature of acetone. We have begun to test the merit of starting freeze substitution at lower temperatures (-140[unreadable]C) with a combination of low-melting solvents in an effort to prevent ice crystal formation at the onset of freeze substitution.